The versatility of proteins makes it possible for complex cellular functions to proceed smoothly, reliably and at a level of efficiency much envied by chemists. A vast array of chemical transformations and molecular recognition phenomena play key roles in the life processes and tremendous opportunities are now becoming apparent in employing the sophisticated protein scaffold and machinery for tailor-made purposes. Designed enzymes, ligands and receptors as well as molecular devices can be envisioned to have a wide spectrum of applications in chemistry, biomedicine and biotechnology.
The functional richness of proteins, based on artificial as well as the naturally occurring amino acids, is now beginning to be explored in designed proteins for purposes of catalysis and binding [W. F. DeGrado, C. M. Summa, Annu. Rev. Biochem. 1999, 68, 779-819; L. Baltzer, Curr. Opin. Struct. Biol 1998, 8, 466-470; C. Micklatcher, J. Chmielewski, Curr. Opin. Chem. Biol. 1999, 3, 724-729; L. Baltzer, J. Nilsson, Curr. Opin. Biotechnol. 2001, 12, 355-360]. The magnitude of the available binding energy that arises from charge-charge, hydrogen bonding and hydrophobic interactions in aqueous solution [A. Fersht, W. H. Freeman and Company, New York, 1999] as well as the capacity of polypeptides for forming a wide range of well-defined tertiary structures make proteins unrivalled molecular scaffolds for probing and exploiting molecular recognition and interactions.
Functional diversity beyond that of folded linear sequences is created in native proteins by enzyme-mediated posttranslational modifications, where site-specific phosphorylations [D. J. Sweatt, Curr. Biol. 2001, 11, R:391-R:394; T. Hunter, Cell 2000, 100, 113-127] and glycosylations [R. A. Dwek, Chem. Rev. 1996, 96, 683-720; H. Lis, N. Sharon, Eur. J. Biochem. 1993, 218, 1-27] are key events in signal transduction, energy storage, immune responses and protein folding. The understanding of how to functionalize folded proteins by controlled reactions would enhance also the repertoire of designed proteins but chemical methods for site-selective functionalization of folded proteins have significant limitations. Substituted thiols can be incorporated if there are Cys residue in the sequence, but if there is more than one cysteine residue the sites of incorporation are statistically controlled and site selectivity is not achieved. Lack of site selectivity characterizes all methods for protein labeling in classical protein chemistry [A. Fersht, W. H. Freeman and Company, New York, 1999] Chemoselective targeting of artificial amino acids [D. S. Kemp, R. I. Carey, Tetrahedron Letters 1991, 32, 2845-2848; P. E. Dawson, S. B. Kent, J. Am. Chem. Soc. 1993, 7263-7266; G. Tuchscherer, Tetrahedron Lett. 1993, 34, 8419-8422; P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent, Science 1994, 776-779] is an attractive approach particularly in combination with recent advances in protein synthesis through chemical ligation [P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent, Science 1994, 776-779; J. P. Tam, Y. Lu, C. Liu, J. Shao, PNAS 1995, 92, 12485-12489]. Template assisted synthesis of proteins (TASP) combines chemoselectivity and orthogonal protection group strategies with the concept of a peptide scaffold for the formation of designed topologies and self-assembled protein structures [M. Mutter, S. Vuileumier, Angew. Chem. Int. Ed. Engl. 1989, 28, 535-554].
Chemical approaches for the site-selective functionalization of folded proteins based on the reactivities of the naturally occurring amino acids are attractive as they provide opportunities for using the powerful methods of molecular biology for selection and screening. Lessons learnt from the modification of model proteins are likely to be of use in functionalizing biologically relevant proteins that cannot be synthesized. Self-catalyzed reactions are very efficient with regards to the incorporation of functional groups and the cost and effort of introducing expensive substituents are considerably less than by synthetic routes, because the amounts needed in the direct reaction with folded proteins are much smaller. The inventor and co-workers have previously reported on a His-Lys mediated site selective functionalization reaction where the side chains of flanking lysine residues in a designed four-helix bundle protein were acylated at pH 5.9 in aqueous solution upon reaction of the peptide with activated esters [L. Baltzer, A.-C. Lundh, K. Broo, S. Olofsson, P. Ahlberg, J. Chem. Soc., Perkin Trans. 2 1996, 1671-1676; K. Broo, A.-C. Lundh, P. Ahlberg, L. Baltzer, J. Am. Chem. Soc. 1996, 118, 8172-8173; L. Andersson, G. Stenhagen, L. Baltzer, J. Org. Chem. 1998, 63, 1366-1367]. In the first, and rate-limiting step of the reaction the unprotonated form of the histidine attacks the ester to form an acyl intermediate. The acyl group is then transferred to the flanking lysine in a fast intramolecular reaction and an amide is formed at the lysine side chain. If several lysine residues are available then at low pH the ones that flank His residues are acylated, whereas those that are far from His residues remain unmodified. If there is more than one lysine in close proximity to the His residue the site of modification is determined by intramolecular competition between the flanking lysines [L. K. Andersson, G. T. Dolphin, J. Kihlberg, L. Baltzer, J. Chem. Soc., Perkin Trans. 2 2000, 459-464.
Biomolecular supramolecular chemistry in aqueous solution is the cornerstone of the life processes. The self-assembly of linear peptides into folded proteins is the pathway by which complex structures for catalysis and binding are formed that are capable of discrimination between the components of the vast biological pool of biomacromolecules and metabolites with almost perfect precision. In addition to the complexity that arises from the naturally occurring amino acids and the large number of available folding motifs [C. Branden, J. Tooze, Introduction to Protein Structure, Garland Publishing, Inc., New York, 1991, Chapter 2], covalent posttranslational modifications add considerable structural and functional variability [H. Lis, N. Sharon, Eur. J. Biochem. 1993, 218, 1-27; R. A. Dwek, Chem. Rev. 1996, 96, 683-720; T. Hunter, Cell 2000, 100, 113-127; D. J. Sweatt, Curr. Biol. 2001, 11, R:391-R:394]. In spite of the opportunities provided by the diversity of protein scaffolds they have, so far, not been explored by chemists for manmade purposes to any significant degree, probably due to the difficulties encountered in understanding protein folding. Recent advances in de novo protein design [J. W. Bryson, S. F. Betz, H. S. Lu, D. J. Suich, H. X. Zhou, K. T. O'Neil, W. F. DeGrado, Science 1995, 270, 935-941; C. K. Smith, L. Regan, Acc. Chem. Res. 1997, 30, 153-161; B. I. Dahiyat, S. L. Mayo, Science 1997, 278, 82-87; C. Micklatcher, J. Chmielewski, Curr. Opin. Chem. Biol. 1999, 3, 724-729; L. Baltzer, H. Nilsson, J. Nilsson, Chem. Rev. 2001] suggest that new proteins can be designed from scratch and that exciting opportunities are now becoming apparent in chemistry, medicine and biotechnology in designing novel proteins for tailor-made purposes.
The protein scaffold is a versatile building block with well-defined distances and geometries between amino acid residues. In a helical segment the distance between α-carbons is 5.2 and 6.3 A, if the residues are three or four residues apart in the sequence, respectively, and several residues along the face of a helix can be used to form sites of great complexity. Larger motifs that combine several secondary structure elements increase the number of addressable functional sites as well as the range of interresidue distances. The size and complexity of proteins, even small ones, compare favorably with what is currently achievable in organic compounds designed to self assemble, especially in aqueous solution. Designed proteins therefore have the potential to become practically useful vehicles for a large variety of purposes. The difficulties encountered in understanding the so called protein folding problem should not be underestimated but from de novo design of proteins has emerged an increased understanding of the relationship between sequence and structure. Several de novo designed proteins that fold into native-like structures have been reported, together with their high-resolution NMR structures [B. I. Dahiyat, S. L. Mayo, Science 1997, 278, 82-87; M. D. Struthers, R. P. Cheng, B. Imperiali, Science 1996, 271, 342-345; C. Schafmeister, S. LaPorte, L. J. W. Miercke, R. M. Stroud, Nature Struc. Biol. 1997, 4, 1039; A. J. Maynard, M. S. Searle, Chem. Commun. 1997, 1297-1298; R. B. Hill, W. F. DeGrado, J. Am. Chem. Soc. 1998, 120, 1138-1145; J. W. Bryson, J. R. Desjarlais, T. M. Handel, W. F. DeGrado, Protein Science 1998, 7, 1404-1414; T. Kortemme, M. Ramirez-Alvarado, L. Serrano, Science 1998, 281,253-256], to support the conclusion that we now know how to design proteins that approach and even surpass a hundred residues in size.
Native proteins are posttranslationally modified in enzyme-catalyzed reactions with high efficiency [G. R. Krishna, F. Wold, in Proteins: Analysis and Design (Ed.: R. H. Angeletti), Academic Press, 1998, pp. 121-206], but few chemical reactions exist that provide the precision needed for the site-specific functionalization of manmade proteins. In order for chemists to be able to make full use of designed protein scaffolds chemical reactions are needed that make it possible to address site-selectively several positions for the introduction of multiple functions in controlled geometries. Classical protein chemistry provides many reactions capable of addressing, chemoselectively, specified amino acid residue side chains, but without site selectivity. Chemoselective reactions [D. S. Kemp, R. I. Carey, Tetrahedron Letters 1991, 32, 2845-2848; P. E. Dawson, S. B. Kent, J. Am. Chem. Soc. 1993, 7263-7266; G. Tuchscherer, Tetrahedron Lett. 1993, 34, 8419-8422; P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent, Science 1994, 776-779] based on the reactivities of functional groups in artificial amino acid residue side chains show great promise, especially in combination with protein synthesis through chemical ligation [P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent, Science 1994, 776-779; J. P. Tam, Y. Lu, C. Liu, J. Shao, Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12485-12489]. There are, however, advantages in site-selective functionalization strategies that are based on the exclusive use of the naturally occurring amino acids. The availability of molecular biological methods for selection and screening makes it possible to refine structures and functions and self-catalyzed functionalization reactions based on the reactivities of the naturally occurring amino acids are very economical in terms of the cost of introducing new functions. While the solid-phase peptide synthesis of peptides require that any amino acid derivative to be introduced is added in large excess, typically tenths of moles of material, self-catalyzed reactions are readily carried out in high yields at micromolar amounts.
The inventor and co-workers have previously reported on site-selective functionalization reactions of lysine side chains based on the cooperativity of His-Lys pairs in helical sequences [L. Baltzer, A.-C. Lundh, K. Broo, S. Olofsson, P. Ahlberg, J. Chem. Soc., Perkin Trans. 2 1996, 1671-1676]. The present invention relates to strategies for addressing lysine residues directly in four-helix bundle proteins using activated ester substrates. In the former, His residues react with the ester in a two step reaction [K. Broo, A.-C. Lundh, P. Ahlberg, L. Baltzer, J. Am. Chem. Soc. 1996, 118, 8172-8173]. The first and rate limiting step is the formation of an acyl intermediate at the His side chain under the release of the leaving group. In the second step, the acyl group is transferred in a fast intramolecular reaction to form an amide at the side chain of the Lys residue, even at a pH below 6, where Lys side chains are predominantly protonated. The pKa of a solvent-exposed Lys residue in aqueous solution is 10.4 [C. Tanford, Advan. Protein Chem. 1962, 17, 69-165] and the efficiency of the acylation reaction is ensured by intramolecularity and cooperativity between the His and Lys residues. If the His residue is flanked by more than one Lys, intramolecular competition determines which Lys is acylated [K. Broo, M. Allert, L. Andersson, P. Erlandsson, G. Stenhagen, J. Wigström, P. Ahlberg, L. Baltzer, J. Chem. Soc., Perkin Trans. 2 1997, 397-398]. In a helix the Lys four residues towards the C-terminal (i,i+4) from the position of the His (i) is the preferred site of acylation, in comparison with the position three residues towards the N-terminus (i,i−3).